Within biotechnology, one of the most widely used separation methods is chromatography. The term chromatography embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions i.e. partitions between the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase. Separated components emerge in a certain order, depending on their interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
The chromatographic methods suggested up to date are based on different modes of interaction with a target. Thus, for example, in ion-exchange chromatography, the functional groups are permanently bonded ionic groups with their counter ions of opposite charge, while in hydrophobic interaction chromatography (HIC), the interaction between the stationary phase and the component to be separated is based on hydrophobicity. Other chromatographic separation principles are well known to the skilled person in the art.
The stationary phase, also known as the separation matrix, comprises a support, which is commonly a plurality of essentially spherical particles, and ligands coupled to the support. In most separation matrices, the support is porous to allow a larger amount of ligand and consequently more bound target compound in each particle. The support is most often a natural or synthetic polymer and the spherical particles may be produced in a number of different ways. Natural polymers often used for this purpose are the polysaccharides dextran and agarose.
The Spinning Disc technology may be used to form agarose beads. It comprises a rotating disc to which liquids are fed on the centre under suitable conditions and centrifuged off the edge. The disc-edge is toothed in order to create droplets. By use of this technology, droplets of uniform size are created at the edge of the disc. The use of Spinning Disc technology for producing sprays, mists and oils of uniform drop size is an established technique. Walter and Prewett (Walton, W. H.; Prewett, W. C. (1949) Proc. Phys. Soc. B. 62, 341-350) concluded as early as 1949 that the size of spray drops is given approximately by the equation (1)
                    d        =                              3.8            ⁢                                          (                                                      T                    /                    D                                    ⁢                                                                          ⁢                  ρ                                )                                            1                /                2                                              ω                                    (        1        )            whereind=drop diameter, D=disc diameter, ω=angular velocity of disc, T=surface tension of liquid, and ρ=density of liquid.
For separation of biomolecules, by chromatographic and batch-wise procedures, the porosity of the beads is very important. One advantage of polymeric media is the opportunity of pore size variation over broad ranges. A general rule, which is accepted to throughout the literature, is to use media with large pore sizes for large molecules. Mass transfer in these pores is a result of diffusion processes and not of convection. It would be desirable to obtain a porosity with a narrow pore size distribution to obtain a selective medium. In the manufacturing of agarose beads using the Spinning Disc technology, it would be desirable to use a procedure by which the bead porosity could be optimized to exclude molecules exceeding a certain chosen size, for example monoclonal antibodies.